Prostate cancer is the most common cancer diagnosis and the second leading cause of cancer- related death in American men. Standard anti-cancer drugs often fail to control localized prostate cancer, suggesting that new targets and new treatment approaches are needed. After the recent discovery of tumor-initiating cells (or cancer stem cells, CSCs) in all major human cancer types, including prostate cancer, accumulated data revealed that not any cancer cell, but only the pathological pool of CSCs are responsible for tumor initiation, development, invasion and response to treatment in the majority of human cancers. Therefore, CSCs are likely to be the most crucial target for cancer treatment. In our previous studies we have identified and characterized several phenotypic subpopulations of stem-like cells, and obtained their genome-wide and pathway-specific characteristics. We have found that prostate cancer cells with the highest expression of CD133 and CD44 possessed high tumor-initiating and sphere-forming capacities, over-activation of several stem cell-related pathways, and up-regulation of many genes involved in the regulation of stem cell self- renewal, pluripotency, anti-apoptosis and drug resistance. We have determined that several new- generation taxoids not only induced significant cell death and growth inhibition in highly aggressive prostate cancer cells grown as 3D CSC-enriched spheroids, but also significantly altered their stemness state, decreasing an expression of several essential markers of pluripotency, including c- Myc, Oct4 and Sox-2, and a large spectrum of other stem cell-related genes. The main hypothesis of this proposal is that selected new-generation taxoids will be effective against prostate CSCs isolated from a broad spectrum of clinical specimens. One of our goals is to develop and standardize a technically straightforward in vitro prostate CSC model for the evaluation of the CSC-targeted drug effects. We will determine the cellular and genomic alterations induced by new-generation taxoids against functionally significant prostate cancer cells, CSCs, not just bulk tumor cells, under stringent, clinically relevant conditions. We will employ new criteria for evaluation of drug effectiveness, including long-term follow-up, and thorough analysis of the residual tumor tissues and cancer spheroids for the presence of the tumor-driving SCSs. In addition, we will create a bank of characterized prostate CSCs, which will be available to the research community. As a result of this project, a potential novel drug against currently incurable androgen-independent prostate cancer can be identified. PUBLIC HEALTH RELEVANCE: Prostate cancer is one of the most frequently diagnosed and mortal cancers in western countries. Despite effective initial tumor shrinkage induced by standard therapies, it usually relapses and develops into more aggressive, inevitably fatal disease. The limited effectiveness of the standard anti-cancer therapies is currently widely attributed to subpopulation of highly drug-resistant tumor-driving cells present in tumors and referred as cancer stem cells (CSCs). For a vast number of human cancers, it is specific pathological pool of drug-resistant CSCs that is responsible for tumor initiation, development, invasion and response to treatment. Therefore, the CSCs are likely to be the most crucial target in the treatment of cancer, and it is important to focus on the drug molecules specifically active against the CSCs, rather than the bulk tumor cells. This new paradigm in cancer treatment requires development of the new criteria of the drug effectiveness and use of clinically relevant models. Accordingly, we aim at targeting a minor, but yet the most critical population of the tumor-driving prostate cancer cells (CSCs) rather than the majority of the proliferating tumor cells, as commonly used. The prostate CSCs are highly drug-resistant, androgen-independent, and it is them that are primarily responsible for tumor progression to incurable hormone-independent disease. The CSCs will be isolated from a broad spectrum of clinical specimens of prostate carcinoma, representing different stages of the disease, not from the established cell lines with particular characteristics. We will focus predominantly on clinically aggressive, advanced prostate carcinoma cases, which are the most difficult to treat. In contrast to commonly used selection of the single cell surface markers for isolation of tumorigenic fraction of cells, we employ combination of multiple stem cell markers and particular levels of their expression as initial morphological criteria for CSC enrichment, which will be further characterized: functionally and genetically, providing for detailed evaluation of their stem cell- and cancer-related features. The isolated cell phenotypes will be cultured and treated under conditions designed to retain their "stemness" and preclude differentiation to the bulk tumor cells. The effectiveness of the selected drug candidates will be tested under clinically relevant conditions using 3D CSC-enriched prostate cancer spheroids. The proposed study will integrate the translational and clinical research at SBU Medical Center (PI: Galina I. Botchkina, PhD;Pathology;Co- Investigators: Dr. Rahuldev Bhalla, MD;Urology;Dr. Liu, MD, PhD;Pathology) and the Institute of Chemical Biology &Drug Development (Dr. Iwao Ojima, Director). In our preliminary studies, we have firstly developed procedures allowing for isolation, purification and propagation of CSC-like cells from solid tumors, biological fluids and established cancer cell lines, and obtained their genome-wide and pathway-specific characteristics. Fraction of cancer cells with high expression of CD133 and CD44 revealed overactivation of several stem cell- specific signaling pathways and many features typical of the pluripotent embryonic stem cells, including upregulation of the genes involved in self-renewal, anti-apoptotic defense and drug resistance as compared to the bulk tumor cells. Secondly, we have found that one of the new generation taxoids, SB-T-1214, not only induced profound cell death and growth inhibition in prostate and colon CSC-enriched spheroids grown in 3D culture systems, but importantly, it significantly attenuated their "stemness" state, decreasing expression of several essential markers of pluripotency and downregulating a large spectrum of other stem cell-related genes. The main objective of this proposal is to find out whether SB-T-1214 or other new-generation taxoids may be efficient against prostate CSCs isolated from a broad spectrum of clinically aggressive prostate carcinoma specimens, thereby potentially providing a long-term cure of the disease. We aim at developing a robust, technically straightforward in vitro prostate CSCs model for evaluation of the CSC-targeted drug effecttiveness under stringent, clinically relevant conditions. Analysis of the molecular and genomic abnormalities of the prostate CSCs in the course of the project will be of obvious usefulness for identification of the new molecular targets for further rational drug design and development of the CSC-targeted anti-cancer therapies.